Adipic acid, i.e., 1,4-butanedicarboxylic acid; COOH(CH2)4COOH, is among the most-produced chemicals worldwide, with approximately 2.5 billion kilograms synthesized annually and a global market of 8 billion USD. The most typical use for adipic acid is for the synthesis of nylon-6,6 used in upholstery, auto parts, apparel, and other products. Standard industrial methods for adipic acid synthesis are costly and have major drawbacks including consumption of fossil fuels, inefficient yields, and production of greenhouse gases. To address this need, “greener” methods for adipic acid production have been demonstrated, but these methods have not been widely adopted, in part because they depend on large-scale hydrogen peroxide oxidation, or because they couple otherwise environmentally friendly fermentation reactions with non-biological synthetic reactions. Sato et al. Science 281: 1646-1647 (1998); Niu et al, Biotechnol. Prog. 18: 201-211 (2002).
A biological method for adipic acid synthesis by a series of recombinant enzymes in bacteria has been discussed. Burgard et al, U.S. Pat. No. 7,799,545; Burgard et al., U.S. Pat. App. Publication Nos. US 2009/0305364; US 2010/0330626; and US 2011/0195466. This biosynthetic pathway has the potential to lower the amounts and costs of input materials, reduce the need for fossil fuel substrates, and reduce the release of pollutants. In this method, genes encoding glutamate-fermenting enzymes from Clostridia symbiosum are expressed in a suitable bacteria such as Escherichia coli to convert 2-oxoadipate, via (R)-2-hydroxyadipate, (R)-2-hydroxyadipoyl-CoA, 2-hexenedioyl-CoA, and 2-hexendioic acid, to adipic acid (FIG. 1A). Parthasarathy et al., Biochemistry 50: 3540-3550 (2011). The compound, 2-oxoadipate (i.e., 2-oxohexanedioic acid), is a natural metabolic intermediate in several organisms (i.e., in lysine catabolism) and experiments show that adipate can be synthesized in bacteria supplied with glucose. Goh et al., Mol. Genet. Metab. 76: 172-180 (2002). In other examples, glutamate is converted to glutarate via (R)-2-hydroxyglutarate, (R)-2-hydroxyglutaryl-CoA), (E)-glutaconyl-CoA), (E)-glutaconate (i.e., (E)-pentenedioic acid), and glutarate, (i.e., pentanedioic acid).
Although other biosynthetic methods have become technically possible, an enzyme for efficient conversion of 2-oxoadipate to (R)-2-hydroxyadipic acid has not been established. Acidaminococcus fermentans 2-hydroxyglutarate dehydrogenase (HghH) was suggested as an enzyme for the conversion of 2-oxoadipate to (R)-2-hydroxyadipate (i.e., (R)-2-hydroxyhexanedioate). Parthasarathy et al., Biochemistry 50: 3540-3550 (2011). However, a drawback of HghH and of 2-hydroxyglutarate dehydrogenases, in general, is that their native substrate is 2-oxoglutarate, the 5-carbon analog of 2-oxoadipate. Consequently, conversion of 2-oxoadipate by HghH is performed 20-times less efficiently than 2-oxoglutarate. Thus, an enzyme that specifically converts 2-oxoadipate to (R)-2-hydroxyadipate, termed an (R)-2-hydroxyadipate dehydrogenase, is needed to overcome the inefficient 2-oxoadipate catalysis and undesired 2-oxoglutarate catalysis associated with HghHs. While 2-hydroxyadipate dehydrogenase activity is observed in nature, the gene that encodes this enzyme has not been identified. Furthermore, this enzyme is not ideal for this method because its substrate specificity is promiscuous and the stereochemistry of the 2-carbon of the product is unknown. Suda, et al., Arch. Biochem. Biophys. 176(2): 610-620 (1976); Suda et al., Biochem. Biophys. Res. Comm. 77(2): 586-591 (1977); Suda et al. Pediatric Res. 12(4): 297-300 (1978).
Isocitrate dehydrogenases (IDHs) are β-hydroxyacid oxidative decarboxylases that convert isocitrate to 2-oxoglutarate (α-ketoglutarate) (FIG. 1B) and are ubiquitous throughout life. Northrop and Cleland, J. Biol. Chem. 249: 2928-2931 (1974); Uhr et al., J. Biol. Chem. 249: 2920-2927 (1974).
Homoisocitrate dehydrogenases (HIDHs) are β-hydroxyacid oxidative decarboxylases from the same subfamily as IDHs that convert homoisocitrate, the 7-carbon analog of isocitrate, to 2-oxoadipate (2-oxohexanedioic acid) (FIG. 1D). HIDHs are involved in an alternative lysine synthetic pathway in yeasts, thermophilic bacteria, and archaea. Miyazaki et al. J. Biol. Chem. 278: 1864-1871 (2003); Xu et al., Cell Biochem. Biophys. 46: 43-64 (2006). Recent exome sequencing revealed missense mutations in NADP+-dependent IDHs that mutate an arginine residue responsible for contacting the β-carboxyl of isocitrate during catalysis. Yan et al., N. Engl. J. Med. 360: 765-773 (2009); Mardis et al., N. Engl. J. Med. 361: 1058-1066 (2009). These mutations cause IDH enzymes to lose their native isocitrate dehydrogenase activity and to gain a neomorphic activity to convert 2-oxoglutarate (α-ketoglutarate) to 2-hydroxyglutarate (2-hydroxypentanedioate) (FIG. 1C). Dang et al., Nature 462: 739-744 (2009); Ward et al., Cancer Cell 17, 225-234 (2010).
The compound 2-hydroxyglutarate is a small biochemical of current interest due to its association with cancer and inborn errors of metabolism. It is of interest to detect and quantify this compound, especially in an enantioselective fashion (i.e., to discriminate the (R)-enantiomer from (S)-2-hydroxyglutarate). This would be useful for research or diagnostics for cancer and inborn errors of metabolism. Mass spectrometry is currently used quantify this compound but this type of instrumentation is specialized and expensive. Therefore, a more accessible quantification method would be useful.
An enzyme that links (R)-2-hydroxyglutarate to NAD+/NADH would allow the development of an NADH-linked assay to quantify (R)-2-hydroxyglutarate. The principle behind this assay would be to add a sample with an unknown amount of (R)-2-hydroxyglutarate to a reaction mix containing a (R)-2-hydroxyglutarate dehydrogenase and NAD+. Then, the (R)-2-hydroxyglutarate dehydrogenase enzyme would convert an equal amount of (R)-2-hydroxyglutarate and NAD+ stoichiometrically to NADH and 2-oxoglutarate. The amount of NADH, which is exactly equal to the amount of input (R)-2-hydroxyglutarate in the sample, can then be measured by UV absorbance (e.g., 340 nm) or fluorescence (e.g., 340 nm excitation; 450 nm emission), or be detected by converting a secondary probe such as resazurin. This type of “enzyme-linked colorimetric assay” scheme is already in place for numerous common biochemicals such as glucose, glutamate, and so forth. This would be useful to lower the cost of (R)-2-hydroxyglutarate quantification, which currently requires mass spectrometry. It could be implemented in research labs, or even provide a diagnostic test in a clinical setting by measuring (R)-2-hydroxyglutarate in tumors, tissue samples, blood, and so forth.
HIDHs from the yeast S. cerevisiae and the thermophilic bacteria T. thermophilus have been studied. Miyazaki et al. J. Biol. Chem. 278: 1864-1871 (2003); Lin et al., Biochemistry 46: 890-898 (2007); Lin et al., Biochemistry 47: 4169-4180 (2008); Lin et al., Biochemistry 48: 7305-7312 (2009); Aktas and Cook, Biochemistry 48: 3565-3577 (2009).
Because IDHs and HIDHs are homologous and functionally related, analogous mutations to HIDHs can cause them to lose their native HIDH activity and to gain the ability to convert 2-oxoadipate to (R)-2-hydroxyadipate (FIG. 1E). Mutations to active site residues of other β-hydroxyacid oxidative decarboxylases can convert these enzymes to 2-hydroxyacid dehydrogenases. That is, instead of catalyzing the removal of a 3-carboxyl group and oxidation of a 2-alcohol group from a substrate to generate a 2-ketone product, the mutants instead catalyze reduction of the same 2-ketone product to the corresponding 2-alcohol. The enzymes also catalyze the reverse reaction (i.e., the oxidation of a 2-alcohol to a 2-ketone).
Alignments of human IDH1 or IDH2 and homoisocitrate dehydrogenases have been performed that show apparent homology among these enzymes. See Aktas and Cook, Biochemistry 48: 3565-3577 (2009). However, correct alignment of these proteins is not trivial. For example, Aktas and Cook incorrectly aligned human IDH1. See Aktas and Cook, FIG. 3 at 3569. The fourth entry in the alignment, Human_ICDH_NADP (i.e., HsIDH1), is not aligned correctly; the sequence should be shifted 8-residues to the right. This mistake was discovered when comparing Human_ICDH_NADP and S. cerevisiae_HICDH in FIG. 3 from Aktas and Cook.
In the correct alignment, the functionally critical residues are aligned with each other (see FIGS. 2B and 3). Residue HsIDH1-R132 is aligned with ScHIDH-R143. HsIDH1-R100, -R109, and -R132, which are important for substrate binding, and -Y139, which is essential for catalysis are aligned with ScHIDH-R114, -R124 and -R143, and Y150, respectively. In contrast, the alignment of Aktas and Cook aligned HsIDH1-R132 with a gap between E132 and K133 in the ScHIDH sequence. This alignment is also incorrect because HsIDH1-G148 was aligned with ScHIDH-R143. It is unlikely that the critical arginine residue could be replaced by a glycine. In addition, there is a conserved branched chain amino acid (e.g., Ile or Leu) before HsIDH1-R100 and ScHIDH-R114 and there are six intervening amino acids between the critical catalytic arginine and tyrosine residues, i.e., HsIDH1-R132/ScHIDH-R143 and HsIDH1-R139/ScHIDH-Y150. Moreover, the experimental evidence described herein robustly supports the alignment in FIGS. 2B and 3. Mutations to positionally aligned residues such as HsIDH1-R132H and ScHIDH-R143H have analogous functional changes. These examples demonstrate that the correct alignment of homologous residues in the IDH and HIDH sequences, inter alia, is unpredictable and requires experimental verification.
Described herein are mutations to residues of the HIDH active site responsible for creating a (R)-2-hydroxyadipate dehydrogenase enzyme (i.e., oxidoreductase) that catalyzes the conversion of 2-oxoadipate to (R)-2-hydroxyadipate. The method used to create such mutants has also been performed for a variety of HIDH enzymes from multiple species. Unique nucleotide and protein sequences were generated using the methods described herein. The method for generating these enzyme constructs was confirmed by biochemical assays that showed catalytic activity in the HIDH mutants (in this case, 2-hydroxyadipate dehydrogenase activity). The HIDH mutants were incorporated into vectors to generate a non-natural microbial organism (e.g., Saccharomyces cerevisiae, yeast). The transformed yeast can be used for the conversion of 2-oxoadipate to commercially useful (E)-2-hexenedioic acid and/or adipic acid products from the metabolism of that organism.
Isopropylmalate dehydrogenases (IPMDHs) and tartarate dehydrogenases (TDHs) are also β-hydroxyacid oxidative decarboxylases that can be mutated to change the activity using the methods described herein. Mutant IPMDHs reduce 4-methyl-2-ketopentanoate to 4-methyl-2-hydroxypentanoate. Mutant TDHs reduce 3-hydroxy-2-oxopropanoic acid (β-hydroxypyruvic acid) to 2,3-dihydroxypropanoic acid.